An enzyme sensor has been developed for the on-line monitoring of penicillin V during its production by fermentation. The enzyme is crosslinked as a very fine film to the sensitive end of a pH transducer, using the spraying method. The biosensor is incorporated in a flow injection analysis (FIA) system within a home-made stirred flow detection cell. This new and improved biosensor shows good stability and large concentration measuring range. Penicillin-V in fermentation broth is detected during the whole HPLC method. On-line measurements are achieved through the automation of the FIA system.